Chaperone-assisted production of active human Rab8A GTPase in Escherichia coli

Protein Expression and Purification
Nathalie BleimlingAymelt Itzen

Abstract

The guanine nucleotide binding protein Rab8A controls the final steps of exocytosis in mammalian cells. It has been implicated in the regulation of apical protein localization in intestinal epithelial cells and ciliary biogenesis. The in vitro structural and biochemical characterization of Rab8A and its interaction with regulator and effector molecules has been hampered by its insolubility in Escherichia coli expression systems. The conventional refolding procedure is laborious and yields only minute amounts of C-terminally truncated Rab8A (Rab8A(1-183): amino acids 1-183), not the full-length protein. Here, we report a method of expressing soluble, hexahistidine-tagged full-length human Rab8A from E. coli. The Rab8A gene was codon-optimized and coexpressed with bacterial GroEL and GroES chaperones. After two-step purification by Ni(2+) affinity chromatography and gel filtration, Rab8A was obtained at a yield of 4 mg protein per 1L of bacterial cell culture and a purity of >95%. The resultant protein was functionally active, as determined by GTPase activity and its interaction with the nucleotide exchange factor MSS4.

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Citations

Mar 14, 2012·Proceedings of the National Academy of Sciences of the United States of America·Lena K OesterlinAymelt Itzen
Jan 6, 2012·Molecular Biology of the Cell·David E Hokanson, Anthony P Bretscher
Jan 5, 2011·Biological Chemistry·Viktor WixlerAymelt Itzen
Jul 2, 2014·Neurobiology of Disease·Guowei YinMarkus Zweckstetter
Jun 23, 2010·Protein Expression and Purification·Sherwin J AbrahamVadim Gaponenko
Jun 24, 2016·ACS Chemical Biology·Philipp M CrommTom N Grossmann
Oct 17, 2015·The EMBO Journal·Yu-Chiang LaiMiratul Mk Muqit
Jun 6, 2018·Frontiers in Cellular and Infection Microbiology·Mathilde M CosséAgathe Subtil
Aug 23, 2020·Nature Communications·Amrita RaiRoger S Goody

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